Method to prepare compositions comprising yeast treated with electromagnetic energy

ABSTRACT

The present invention relates to pharmaceutical compositions and dietary supplement comprising yeast cells that can produce a healthful benefit in a subject inflicted with pancreatic cancer. The biological compositions can be used to retard the growth of pancreatic cancer cells and/or prolonging the time of survival of the subject. The invention also relates to methods for manufacturing the biological compositions.

1. FIELD OF THE INVENTION

The invention relates to oral compositions comprising yeast cells thatcan produce a healthful benefit in a subject inflicted with pancreaticcancer. The invention also relates to methods for manufacturing the oralcompositions and methods of use thereof.

2. BACKGROUND OF THE INVENTION

2.1 Pancreatic Cancer

Pancreatic cancer is the second most common visceral malignancy as wellas the fifth leading cause of cancer mortality in the United States,accounting for one fifth of all gastrointestinal (GI) cancer deaths.Pancreatic cancer is a disease of the industrialized world. There is atenfold difference between the highest incidence rate, in American blackmales (15.2 per 100,000), and the lowest rates, in Hungary, Nigeria, andIndia (1.5 per 100,000) (Waterhouse et al., 1976, Lyon: InternationalAgency for Research on Cancer, Vol. 3). High risk has also been observedin Polynesian males, including native Hawaiians and New Zealand Maoris.Like many other cancers, pancreatic cancer usually strikes after age 50.The incidence of pancreatic cancer has risen with an increase in theaverage life span. For example, the incidence in Japan rose from 1.8 per100,000 in 1960 to 5.2 per 100,000 in 1985 (Beazley et al., 1995,Clinical Oncology, Chapter 15).

Aside from advanced age, smoking is the main risk factor for pancreaticcancer—a smoker is three to four times more likely than a nonsmoker toacquire the disease. People frequently exposed to certain petroleum,chemical and metal products may also be at increased risk. Excessivedietary fat and protein as well as low fiber intake may promote thedisease. Diabetes is linked to pancreatic cancer for 10% to 20% ofpatients diagnosed with pancreatic cancer also have diabetes. Otherhereditary diseases associated with pancreatic cancer includeinflammatory pancreatic problem, Gardner's syndrome (where growthsdevelop inside and outside the colon), the skin and nerve diseaseneurofibromatosis, and multiple endocrine neoplasia, a condition thatpromotes growth of noncancerous islet cell

Pancreatic cancer is difficult to detect early because the pancreas islocated deep inside the body and is hidden behind other organs. Theretroperitoneal location of the pancreas is considered a major obstacleto early treatment. Further, pancreatic cancer does not usually causesymptoms in its early stages. Even if symptoms do occur, they may bevague and the tumor has already spread outside of the pancreas(metastasis). Signs include abdominal pain, unexpected weight loss,nausea, loss of appetite, weight loss, digestive problems, jaundice, oryellowing of the skin are nonspecific and often overlap with otherdiseases. The rarer endocrine (or islet cell) cancers may also causerestlessness, loss of energy, irritability, sweating, tremor, drowsinessand severe confusion. Because the symptoms are so general in nature,several diagnostic tools are frequently used, e.g., ultrasound, CT scan,MRI (magnetic resonance imaging), barium meal ERCP (endoscopicretrograde cholangiopancreatography) and PTC (percutaneous transhepaticcholangiopancreatography) tests.

The staging of pancreatic cancer is based on the revised criteria of TNMstaging by the American Joint Committee for Cancer (AJCC) published in1988. Staging is the process of describing the extent to which cancerhas spread from the site of its origin. It is used to assess a patient'sprognosis and to determine the choice of therapy. The stage of a canceris determined by the size and location in the body of the primary tumor,and whether it has spread to other areas of the body. Staging involvesusing the letters T, N and M to assess tumors by the size of the primarytumor (T); the degree to which regional lymph nodes (N) are involved;and the absence or presence of distant metastases (M)—cancer that hasspread from the original (primary) tumor to distant organs or distantlymph nodes. Each of these categories is further classified with anumber 1 through 4 to give the total stage. Once the T, N and M aredetermined, a “stage” of I, II, III or IV is assigned. Stage I cancersare small, localized and usually curable. Stage II and III cancerstypically are locally advanced and/or have spread to local lymph nodes.Stage IV cancers usually are metastatic (have spread to distant parts ofthe body) and generally are considered inoperable.

More than 90% of pancreatic malignancies arise from ductal epithelium,even though less than 15% of the pancreas by mass is made up of ductaltissue. At least 90% of pancreatic cancers are exocrine cell cancerscalled adenocarcinomas, usually originating in the head of the gland.Other tumors arising from the pancreas include acinar cell carcinoma(about 5%), cystadenocarcinoma (mucinous), adenosquamous carcinoma,solid microglandular carcinoma, carcinoid, sarcoma, and malignantlymphoma. Cancers arising in the head of the pancreas must bedistinguished from peripancreatic lesions arising from the distal commonbile duck, the ampulla of Vater, or the duodenum. While ampullary canceris the most resectable and associated with the most favorable prognosis,survival rates with all three are higher than with pancreatic cancer.

Surgery is by far the most effective choice of treatment. A Whippleprocedure removes the head of the pancreas, part of the small intestine,and some of the tissues around it. Enough of the pancreas is left tocontinue making digestive juices and insulin. Total pancreatectomy takesout the whole pancreas, part of the small intestine, part of thestomach, the bile duct, the gallbladder, spleen, and most of the lymphnodes in the area. Distal pancreatectomy takes out only the tail of thepancreas. Unfortunately, roughly 80% of individuals on presentation areineligible for a curative attempt once metastasis to the peritonealsurfaces, the omentum, the liver, and the transverse mesocolon takesplace. At that point, surgery is performed to relieve symptoms. Forinstance, if the cancer is blocking the small intestine and bile buildsup in the gallbladder, biliary bypass may be performed by sewing thegallbladder or bile duck directly to the small intestine.

Further, Whipple procedure, although a popular choice of treatment, cancause numerous complications such as sepsis, biliary or pancreaticfistula, and bleeding. The overall morbidity rate varies between 27% and46%. (Pellegrini et al., 1989, Arch Surg. 124:778–81). Until recently,5-year survival after pancreaticoduodenectomy for cancer was exceedinglyrare. Survival rates of 3% to 25% are currently being reported.Occasionally, long-term survivors are reported with a large tumor, butthe majority of the survivors are those who have small lesions andnegative lymph nodes (T1, N0, M0). Mean survival after pancreaticresection is 17 months.

The use of biological therapy (using the body's immune system to fightcancer), otherwise known as biological response modifier (BRM) therapyor immunotherapy, is currently being tested for pancreatic cancer.Similarly, adjuvant therapy is gaining popularity by administering tothe patient after “successful” Whipple resections, either 5-fluorouracil(5-FU) or radiation therapy (20 Gy). Studies have reported that themedian survival for the control groups was 11 months, compared with 20months for the treatment groups (Kalser et al., 1985, Arch Surg.120:899–903).

Although initially considered to be radioresistant, pancreatic exocrinetumors are responsive to radiation therapy. Nonetheless, the curativerate is extremely low and the side effects are undesirable. Theproximity of radiosensitive tissues, including the liver, kidney, andduodenum, severely limits the efficacy of external-beam radiationtherapy and has led to innovative approaches for delivering high-dosetreatment, including precision high-dose external-beam techniques,interstitial or brachytherapy, and intraoperative and adjuvant radiationtherapy. There is reported value for all these techniques, but becauseof the heterogeneity of patients, tumor size, stage of disease,performance status, and volume of the tumor, it is difficult to make adefinitive conclusion concerning the superiority of one modality overthe others. Titanium clips, which do not interfere with CT studies, canbe surgically placed to mark the margins of the tumor, thus enabling theradiation therapist to design fields that will maximize tumor dosage andminimize injury to radiosensitive, normal adjacent structures(Dodelbower et al., 1984, World J Surg. 8:919–28).

Many chemotherapeutic drugs have been tried in the past as single agentsfor the palliation of pancreatic cancer, but the results were generallydisappointing. Nevertheless, the role of chemotherapy in the managementof pancreatic cancer is continually evolving. Oftentimes, chemotherapywith radiation in adjunct to surgery is used. In general, chemotherapycan achieve long-term survival rates of up to 15% to 20%, even inpatients with recurrent or metastatic disease (Ali et al., 2000,Oncology 14(8):1223–30). Unfortunately, the high initial response ratesto first line chemotherapy does not appear to translate into a survivalbenefit (Kohno and Kitahara, 2001, Gan To Kagaku Ryoho 28(4):448–53).Moreover, there are many undesirable side effects associated withchemotherapy such as temporary hair loss, mouth sores, anemia (decreasednumbers of red blood cells that may cause fatigue, dizziness, andshortness of breath), leukopenia (decreased numbers of white blood cellsthat may lower resistance to infection), thrombocytopenia (decreasednumbers of platelets that may lead to easy bleeding or bruising), andgastrointestinal symptoms like nausea, vomiting, and diarrhea. Activechemotherapeutic agents include hexamethylmelamine, bleomycin,cisplatin, mitomycin C, doxorubicin, methotrexate and Gemzar(gemcitabine HCL).

The identification of active chemotherapeutic agents against cancerstraditionally involved the use of various animal models of cancer. Themouse has been one of the most informative and productive experimentalsystem for studying carcinogenesis (Sills et al., 2001, Toxicol Letters120:187–198), cancer therapy (Malkinson, 2001, Lung Cancer32(3):265–279; Hoffman R M., 1999, Invest New Drugs 17(4):343–359), andcancer chemoprevention (Yun, 1999, Annals NY Acad Sci. 889:157–192).Cancer research started with transplanted tumors in animals whichprovided reproducible and controllable materials for investigation.Pieces of primary animal tumors, cell suspensions made from thesetumors, and immortal cell lines established from these tumor cellspropagate when transplanted to animals of the same species.

To transplant human cancer to an animal and to prevent its destructionby rejection, the immune system of the animal are compromised. Whileoriginally accomplished by irradiation, thymectomy, and application ofsteroids to eliminate acquired immunity, nude mice that are athymiccongenitally have been used as recipients of a variety of human tumors(Rygaard, 1983, in 13^(th) International Cancer Congress Part C, Biologyof Cancer (2), pp37–44, Alan R. Liss, Inc., NY; Fergusson and Smith,1987, Thorax, 42:753–758). While the athymic nude mouse model providesuseful models to study a large number of human tumors in vivo, it doesnot develop spontaneous metastases and are not suitable for all types oftumors. Next, the severe combined immunodeficient (SCID) mice isdeveloped in which the acquired immune system is completely disabled bya genetic mutation. Human lung cancer was first used to demonstrate thesuccessful engraftment of a human cancer in the SCID mouse model (ReddyS., 1987, Cancer Res. 47(9):2456–2460). Subsequently, the SCID mousemodel have been shown to allow disseminated metastatic growths for anumber of human tumors, particularly hematologic disorders and malignantmelanoma (Mueller and Reisfeld, 1991, Cancer Metastasis Rev.10(3):193–200; Bankert et al., 2001, Trends Immunol. 22:386–393). Withthe recent advent of transgenic technology, the mouse genome has becomethe primary mammalian genetic model for the study of cancer (Resor etal., 2001, Human Molec Genet. 10:669–675).

While surgery, chemotherapeutic agents, hormone therapy, and radiationare useful in the treatment of pancreatic cancer, there is a continuedneed to find better treatment modalities and approaches to manage thedisease that are more effective and less toxic, especially when clinicaloncologists are giving increased attention to the quality of life ofcancer patients. The present invention provides an alternative approachto cancer therapy and management of the disease by using an oralcomposition comprising yeasts.

2.2 Yeast-Based Compositions

Yeasts and components thereof have been developed to be used as dietarysupplement or pharmaceuticals. However, none of the prior methods usesyeast cells which have been cultured in an electromagnetic field toproduce a product that has an anti-cancer effect. The following are someexamples of prior uses of yeast cells and components thereof:

U.S. Pat. No. 6,197,295 discloses a selenium-enriched dried yeastproduct which can be used as dietary supplement. The yeast strainSaccharomyces boulardii sequela PY 31 (ATCC 74366) is cultured in thepresence of selenium salts and contains 300 to about 6,000 ppmintracellular selenium. Methods for reducing tumor cell growth byadministration of the selenium yeast product in combination withchemotherapeutic agents is also disclosed.

U.S. Pat. No. 6,143,731 discloses a dietary additive containing wholeβ-glucans derived from yeast, which when administered to animals andhumans, provide a source of fiber in the diet, a fecal bulking agent, asource of short chain fatty acids, reduce cholesterol and LDL, andraises HDL levels.

U.S. Pat. No. 5,504,079 discloses a method of stimulating an immuneresponse in a subject utilizing modified yeast glucans which haveenhanced immunobiologic activity. The modified glucans are prepared fromthe cell wall of Saccharomyces yeasts, and can be administered in avariety of routes including, for example, the oral, intravenous,subcutaneous, topical, and intranasal route.

U.S. Pat. No. 4,348,483 discloses a process for preparing a chromiumyeast product which has a high intracellular chromium content. Theprocess comprises allowing the yeast cells to absorb chromium under acontrolled acidic pH and, thereafter inducing the yeast cells to grow byadding nutrients. The yeast cells are dried and used as a dietarysupplement.

Citation of documents herein is not intended as an admission that any ofthe documents cited herein is pertinent prior art, or an admission thatthe cited documents are considered material to the patentability of theclaims of the present application. All statements as to the date orrepresentations as to the contents of these documents are based on theinformation available to the applicant and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

3. SUMMARY OF THE INVENTION

The present invention relates to biological or oral compositions usefulfor subjects with pancreatic cancer. In one embodiment, the presentinvention provides biological compositions comprising live yeast cellswhich are capable of producing a healthful benefit in subjects withpancreatic cancer. In other embodiments, the invention provides methodsof making the biological compositions, and methods of using thebiological compositions.

In particular, the methods of the invention comprise culturing yeastcells in the presence of a series of electromagnetic fields such thatthe yeast cells becomes metabolically active. The electromagnetic fieldsused are each defined by one of five frequency ranges and a broad rangeof field strength. The starting yeast cells are commercially availableand/or accessible to the public, such as but not limited toSaccharomyces. The methods for making the biological compositions of theinvention further comprise conditioning the activated yeast cells inplant extracts and the gastric juice of animals, while in the presenceof another series of electromagnetic fields.

The methods of manufacturing also comprise expanding the number ofactivated or activated and conditioned yeast cells in large scalecultures in the presence of yet another series of electromagneticfields, performing quality control measures, and packaging.Pharmaceutical compositions of the invention comprises activated andconditioned yeast cells and one or more pharmaceutically acceptableexcipients or carriers. Additional ingredients, such as vitamins and/orflavors may be added to the biological compositions to form the oralcompositions of the invention. Such additional carriers and ingredientscan improve the healthful benefits, pharmacological properties, andorganoleptic characteristics of the oral compositions. During themanufacturing process, the activated or activated and conditioned yeastcells may be dried and stored for a period of time.

The biological or oral compositions of the invention are ingested by thesubject or used as an additive to be incorporated into food to beconsumed by the subject. Dietary supplement and nutritional compositionscomprising activated and conditioned yeast cells are encompassed by theinvention. Preferably, the subject is a human being.

In various embodiments, the biological or oral compositions of theinvention are used to produce a healthful benefit in a subject withpancreatic cancer or at high risk of developing pancreatic cancer. Inparticular, the biological composition of the invention can retard thegrowth of pancreatic cancer cells in an animal which received thecomposition orally. The composition can also be used to prolong the timeof survival of an animal with pancreatic cancer.

4. BRIEF DESCRIPTION OF FIGURES

FIG. 1 Activation and conditioning of yeast cells. 1 yeast cell culture;2 container; 3 electromagnetic field source; 4 electrode.

FIG. 2 Large scale propagation of yeast cells. 5 first container; 6second container; 7 third container; 8 yeast cell cultures; 9electromagnetic field source.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to biological compositions that canproduce a healthful benefit in a subject with pancreatic cancer. Thepresent invention provides methods for manufacturing the biologicalcompositions as well as methods for using the biological compositions.

In one embodiment, the invention provides biological compositions thatcomprise yeasts. Unlike the traditional use of yeasts in the making offood, the yeast cells of the invention are not used as a source ofenzymes that acts on the food ingredients. The yeasts are not a primarysource of nutrients for the subject. Nor are yeast cells used as acarrier, such as metal salts. The yeast cells of the invention are livewhen administered orally or ingested along with food by a subject.Without being bound by any theory or mechanism, the inventor believesthat the culture conditions activate and/or amplified the expression ofa gene or a set of genes in the yeast cells such that the yeast cellsbecomes highly effective in stimulating the animal's immune system,including both specific and non-specific immunological reactions, theresults of which are manifested as the overall healthful benefitsobserved in the treated subject. The healthful benefits provided byusing the biological compositions are demonstrated in animal models ofhuman pancreatic cancer which show inhibition of tumor growth andprolonged survival time of animals with the disease.

In another embodiment, the invention provides methods for making theyeast cells in the biological compositions. The starting materials arenormal yeast cells which can be readily obtained commercially or frompublic microorganism deposits. The methods of the invention comprise aset of culture conditions that can be applied reproducibly to activatethe yeast cells. The key feature of the culture conditions used in themethods of the invention is a series of alternating electromagneticfields of defined frequency ranges and field strengths which are appliedto the growing yeast cell culture. The method further comprises the stepof conditioning the activated live yeast cells to the acidic environmentof the stomach of the subject. The electromagnetic fields used in thesemethods can be created reproducibly at various scales, thus enablingeven the large scale manufacturing of the biological compositions of theinvention. By careful control of the culturing conditions, normal yeastcells can be activated routinely and reproducibly to become yeast cellsof the invention.

In yet another embodiment, the invention provides methods formanufacturing an oral composition comprising activated and conditionedyeasts of the invention, and additional ingredients, including but notlimited to pharmaceutically acceptable carriers or excipients, vitamins,herbs (including traditional Chinese medicine products), herbalextracts, minerals, amino acids, flavoring agents, coloring agents,and/or preservatives.

In yet another embodiment, the biological compositions can be added tofood which will be consumed by the subject. As known to those skilled inthe relevant art, many methods may be used to mix the biological or oralcompositions of the invention with food while the yeast cells remainviable. In a particular embodiment, the culture broth comprising liveyeast cells of the present invention are added directly to food justprior to consumption. Dried powders of the yeasts can also bereconstituted and added directly to food just prior to consumption.

In various embodiments, the oral compositions of the invention can beconsumed directly by a subject or be fed directly to a subject. Forexample, the subject may drink the culture broth or a fraction thereofthat comprises live activated and conditioned yeast cells. Oralcompositions comprising dried yeast cells can also be given as a soliddosage form to the subject.

Although it is not necessary, the biological or oral compositions of theinvention can be used in conjunction or in rotation with other types oftreatment modalities such as but not limited to surgery,chemotherapeutic agents, and radiation. Since the biologicalcompositions of the invention are administered orally, the assistance ofhealth professionals in administration of the composition is generallynot essential.

Described below in Section 5.1 are the yeast cells of the invention andmethods of their preparation. Section 5.2 describes the use of thebiological compositions of the invention in a subject suffering frompancreatic cancer. The examples in Sections 6 to 9 demonstrate thetherapeutic benefits of an oral composition of the invention. Theactivated and conditioned yeast cells in the oral composition arecharacterized by their ability to (i) suppress the growth of cancercells in an animal model of human pancreatic cancer, or (ii) prolong thesurvival of animals with transplanted cancer cells in a model of humanpancreatic cancer, as compared to yeast cells which have not beenactivated and conditioned.

5.1 Preparation of the Yeast Cell Cultures

The yeast cells of the biological composition are produced by culturinga plurality of yeast cells in an appropriate culture medium in thepresence of an alternating electromagnetic field over a period of time.The method comprises a first step of activating the yeast cells and asecond step of conditioning the activated yeast cells. The activationprocess comprises culturing yeast cells in the presence of at least two,three, four or five electromagnetic fields of specific frequencies andfield strength. The conditioning process comprises further culturing ofthe activated yeast cells in a medium comprising plant extracts andextracts from the stomach of an animal, in the presence of at least oneelectromagnetic field. The activated and conditioned yeast cells can bestored as dried cells after drying the cells under appropriateconditions. The dried activated and conditioned yeast cells can be usedlater in large scale culturing processes for manufacturing thebiological compositions of the invention. The various culturingprocesses of the invention can be performed either as a batch process ora continuous process.

5.1.1 Yeasts

In various embodiments, yeasts of the genera of Saccharomyces, Candida,Crebrothecium, Geotrichum, Hansenula, Kloeckera, Lipomyces, Pichia,Rhodosporidium, Rhodotorula, Torulopsis, Trichosporon, and Wickerhamiacan be used in the invention. Generally, fungi used for foodmanufacturing are preferred.

Non-limiting examples of yeast strains include Saccharomyces sp.,AS2.311; Schizosaccharomyces pombe Linder, AS2.214, AS2.248, AS2.249,AS2.255, AS2.257, AS2.259, AS2.260, AS2.274, AS2.994, AS2.1043,AS2.1149, AS2.1178, IFFI 1056; Saccharomyces sake Yabe, ACCC2045;Saccharomyces uvarum Beijer, IFFI 1023, IFFI 1032, IFFI 1036, IFFI 1044,IFFI 1072, IFFI 1205, IFFI 1207; Saccharomyces rouxii Boutroux, AS2.178,AS2.180, AS2.370, AS2.371; Saccharomyces cerevisiae Hansen Var.ellipsoideus, ACCC2043, AS2.2, AS2.3, AS2.8, AS2.53, AS2.163, AS2.168,AS2.483, AS2.541, AS2.559, AS2.606, AS2.607, AS2.611, AS2.612;Saccharomyces carlsbergensis Hansen, AS2.116, AS2.162, AS2.189, AS2.200,AS2.216, AS2.265, AS2.377, AS2.417, AS2.420, AS2.440, AS2.441, AS2.443,AS2.444, AS2.459, AS2.595, AS2.605, AS2.638, AS2.742, AS2.745, AS2.748,AS2.1042; Rhodotorula aurantiaca (Saito) Ladder; AS2.102, AS2.107,AS2.278, AS2.499, AS2.694, AS2.703, AS2.704 and AS2.1146; Saccharomycescerevisiae Hansen, ACCC2034, ACCC2035, ACCC2036, ACCC2037, ACCC2038,ACCC2039, ACCC2040, ACCC2041, ACCC2042, AS2.1, AS2.4, AS2.11, AS2.14,AS2.16, AS2.56, AS2.69, AS2.70, AS2.93, AS2.98, AS2.101, AS2.109,AS2.110, AS2.112, AS2.139, AS2.173, AS2.182, AS2.196, AS2.242, AS2.336,AS2.346, AS2.369, AS2.374, AS2.375, AS2.379, AS2.380, AS2.382, AS2.393,AS2.395, AS2.396, AS2.397, AS2.398, AS2.399, AS2.400, AS2.406, AS2.408,AS2.409, AS2.413, AS2.414, AS2.415, AS2.416, AS2.422, AS2.423, AS2.430,AS2.431, AS2.432, AS2.451, AS2.452, AS2.453, AS2.458, AS2.460, AS2.463,AS2.467, AS2.486, AS2.501, AS2.502, AS2.503, AS2.504, AS2.516, AS2.535,AS2.536, AS2.558, AS2.560, AS2.561, AS2.562, AS2.576, AS2.593, AS2.594,AS2.614, AS2.620, AS2.628, AS2.631, AS2.666, AS2.982, AS2.1190,AS2.1364, AS2.1396, IFFI 1001, IFFI 1002, IFFI 1005, IFFI 1006, IFFI1008, IFFI 1009, IFFI 1010, IFFI 1012, IFFI 1021, IFFI 1027, IFFI 1037,IFFI 1042, IFFI 1045, IFFI 1048, IFFI 1049, IFFI 1050, IFFI 1052, IFFI1059, IFFI 1060, IFFI 1062, IFFI 1202, IFFI 1203, IFFI 1209, IFFI 1210,IFFI 1211, IFFI 1212, IFFI 1213, IFFI 1215, IFFI 1221, IFFI 1224, IFFI1247, IFFI 1248, IFFI 1251, IFFI 1270, IFFI 1277, IFFI 1289, IFFI 1290,IFFI 1291, IFFI 1292, IFFI 1293, IFFI 1297, IFFI 1300, IFFI 1301, IFFI1302, IFFI 1307, IFFI 1308, IFFI 1309, IFFI 1310, IFFI 1311, IFFI 1331,IFFI 1335, IFFI 1336, IFFI 1337, IFFI 1338, IFFI 1339, IFFI 1340, IFFI1345, IFFI 1348, IFFI 1396, IFFI 1397, IFFI 1399, IFFI 1441 and IFFI1443. Preferred yeast strains include but are not limited to S.cerevisiae AS2.501, AS2.502, AS2.503, AS2.504, AS2.535, AS2.558,AS2.560, AS2.561 and AS2.562.

Generally, yeast strains useful for the invention can be obtained fromprivate or public laboratory cultures, or publicaly accessible culturedeposits, such as the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209 and the China GeneralMicrobiological Culture Collection Center (CGMCC), China Committee forCulture Collection of Microorganisms, Institute of Microbiology, ChineseAcademy of Sciences, Haidian, P.O. Box 2714, Beijing, 100080, China.

Non-limiting examples of using yeast cells of the invention withSaccharomyces cerevisiae Hansen strain IFFI 1413 are provided inSections 6 to 9 herein below. The yeast cells of the invention do notcomprise an enhanced level of selenium or chromium relative to thatfound in naturally occurring yeast cells. In certain embodiments, thebiological compositions do not comprise cells of Saccharomyces boulardii(for example, ATCC Accession No. 74366) or cells of a particular strainof Saccharomyces cerevisiae (strain Hansen CBS 5926) that is alsocommonly referred to as Saccharomyces boulardii.

Although it is preferred, the preparation of the yeast cells of theinvention is not limited to starting with a pure strain of yeast. Theyeast cells in the biological compositions may be produced by culturinga mixture of yeast cells of different species or strains. Theconstituents of a mixture of yeast cells can be determined by standardyeast identification techniques well known in the art.

In various embodiments of the invention, standard techniques forhandling, transferring and storing yeasts are used. Although it is notnecessary, sterile conditions or clean environments are highly desirablewhen carrying out the manufacturing processes of the invention,especially when the biological compositions are for human consumption.The manufacturing process can be adapted to meet regulatory guidelineson product safety and quality control by standard practice known in theart.

5.1.2 Electromagnetic Fields

As used herein, the terms “alternating electromagnetic field”,“electromagnetic field” or “EM field” are synonymous. An electromagneticfield useful in the invention can be generated by various means wellknown in the art. A schematic illustration of exemplary setups aredepicted respectively in FIG. 1. An electromagnetic field of a desiredfrequency and a desired field strength is generated by anelectromagnetic wave source (3) which comprises one or more signalgenerators that are capable of generating electromagnetic waves,preferably sinusoidal waves, and preferably in the frequency range of1,500 to 15,000 MHz and most preferably 7,900 to 12,800 MHz. Such signalgenerators are well known in the art. Signal generators capable ofgenerating signal with a narrower frequency range can also be used. Ifdesirable, a signal amplifier can also be used to increase the outputsignal, and thus the strength of the EM field.

The electromagnetic field can be applied to the culture by a variety ofmeans including placing the yeast cells in close proximity to a signalemitter connected to a source of electromagnetic waves. The signalgenerator is connected to the signal emitter by cables such as coaxialcables that can transmit signals up to greater than or equal to 30 GHz.Typically, the yeast cells are placed in a container which is made ofmaterial that is not an electric conductor, such as but not limited toplastic, resin, glass, and ceramic.

In one embodiment, the electromagnetic field is applied by signalemitters in the form of electrodes (4) that are submerged in a cultureof yeast cells (1). In a preferred embodiment, one of the electrodes isa metal plate which is placed on the bottom of a non-conductingcontainer (2), and the other electrode comprises a plurality of wires ortubes so configured inside the container such that the energy of theelectromagnetic field can be evenly distributed in the culture. Theelectrodes are preferably made of copper. For an upright culture vessel,the tips of the wires or tubes are placed within 3 to 30 cm from thebottom of the vessel (i.e., approximately 2% to 10% of the height of thevessel from the bottom). Table 1 provides exemplary set up for culturingthe yeast cells of the invention.

TABLE 1 height of culture medium in the distance electrodes are rangefor distance of non-conducting placed from the bottom the electrodesfrom container (cm) of the container (cm) the bottom (cm) 15 to 20 3 3to 5 20 to 30 5 5 to 7 30 to 50 7  7 to 10 50 to 70 10 10 to 15  70 to100 15 15 to 20 100 to 150 20 20 to 30 150 to 200 30 25 to 30

The number of electrodes used depends on both the volume of the cultureand the diameter of the electrode. For example, for a culture having avolume of 10 liter or less, two or three electrodes having a diameter ofbetween 0.5 to 2.0 mm can be used. For a culture volume of 10 to 100liter of culture, the electrodes can have a diameter of 3.0 to 5.0 mm.For a culture volume of 100 to 1,000 liter, the electrodes can have adiameter of 6.0 to 15.0 mm. For a culture having a volume greater than1,000 liter, the electrodes can have a diameter of between 20.0 to 25.0mm.

5.1.3 Activation of Yeast Cells

According to the invention, the method for producing activated yeastcells of the invention comprises culturing yeast cells in the presenceof at least two, three, four or five alternating electromagnetic (EM)fields.

The culture process can be initiated by inoculating 1,000 ml of mediumwith an inoculum of a selected yeast strain (such as one of thosedescribed in Section 5.1.1) such that the starting cell density of theculture is greater than about 10⁵ cells per ml. For example,Saccharomyces cerevisiae Hansen strain IFFI 1413 can be used. Thestarting culture can be used to seed larger scale culture. The cultureis maintained initially at 28° C. to 32° C. for 22 to 30 hours prior toexposure to the EM field(s), typically at 30° C. for 28 hours.

The culturing process may preferably be conducted under conditions inwhich the concentration of dissolved oxygen is between 0.025 to 0.08mol/m³, preferably 0.04 mol/m³. The oxygen level can be controlled byany conventional means known in the art, including but not limited tostirring and/or bubbling.

The culture is most preferably carried out in a liquid medium whichcontains sources of nutrients assimilable by the yeast cells. Table 2provides an exemplary medium for culturing the yeast cells of theinvention.

TABLE 2 Medium Composition Quantity Sucrose or glucose 20 g Vitamin B₃50 μg Vitamin B₁₂ 30 μg Vitamin H 20 μg Fetal calf serum 40 ml KH₂PO₄0.20 g MgSO₄.7H₂O 0.25 g NaCl 0.30 g CaSO₄.2H₂O 0.20 g CaCO₃.5H₂O 4.0 gPeptone 2.5 g Autoclaved water 1,000 ml

The culturing medium is heated to 45° C. and cooled before adding thevitamin B₃, vitamin B₁₂, vitamin H, and fetal calf serum.

In general, carbohydrates such as sugars, for example, sucrose, glucose,fructose, dextrose, maltose, xylose, and the like and starches, can beused either alone or in combination as sources of assimilable carbon inthe culture medium. The exact quantity of the carbohydrate source orsources utilized in the medium depends in part upon the otheringredients of the medium but, in general, the amount of carbohydrateusually varies between about 0.1% and 5% by weight of the medium and ispreferably between about 0.2% and 2%. These carbon sources can be usedindividually, or several such carbon sources may be combined in themedium. Among the inorganic salts which can be incorporated in theculture media are the customary salts capable of yielding sodium,calcium, phosphate, sulfate, carbonate, and like ions. Non-limitingexamples of nutrient inorganic salts are KH₂PO₄, (NH₄)₂HPO₄, CaCO₃,MgSO₄, NaCl, and CaSO₄.

It should be noted that the composition of the media provided in Table 2is not intended to be limiting. The process can be scaled up or downaccording to needs. Various modifications of the culture medium may bemade by those skilled in the art, in view of practical and economicconsiderations, such as the scale of culture and local supply of mediacomponents.

In certain embodiments, a series of at least two, three, four or five EMfields are applied to the culture of yeast cells, each having adifferent frequency within a stated range, and a different fieldstrength within a stated range. The EM fields can be applied in anyorder and by any means known in the art, such as the apparatus describedin Section 5.1.2. Although any of the following two, three or four EMfields can be applied, preferably, all five EM fields are applied.

For the first EM field, the frequency is in the range of 7,961 to 7,970MHz and the field strength is in the range of 250 to 270 mV/cm. Theyeast culture is exposed to this first EM field at 30±2° C. for about 22hours.

For the second EM field, the frequency is in the range of 10,181 to10,190 MHz and the field strength is in the range of 250 to 270 mV/cm.The yeast culture is exposed to this second EM field at 30±2° C. forabout 10 hours.

For the third EM field, the frequency is in the range of 12,276 to12,285 MHz and the field strength is in the range of 270 to 290 mV/cm.The yeast culture is exposed to this third EM field at 30±2° C. forabout 24 hours.

For the fourth EM field, the frequency is in the range of 12,461 to12,470 MHz and the field strength is in the range of 280 to 300 mV/cm.The yeast culture is exposed to this fourth EM field at 30±2° C. forabout 10 hours.

For the fifth EM field, the frequency is in the range of 12,761 to12,770 MHz and the field strength is in the range of 300 to 320 mV/cm.The yeast culture is exposed to this fifth EM field at 30±2° C. forabout 18 hours.

In less preferred embodiments, the yeast cells can be cultured byexposure to two, three or four of the above-mentioned EM fields in adifferent order. The yeast cells can remain in the same container anduse the same set of electromagnetic wave generator and emitters whenswitching from one EM field to another EM field.

The cell density of the culture at the end of the activation process istypically greater than about 10⁶ to 10⁹ cells per ml (estimated byhematocytometer). The activated yeast cells may be recovered from theculture by various methods known in the art, and stored at a temperaturebelow about 0° C. to 4° C. The activated yeast cells recovered from theliquid culture may be dried and stored in powder form. Preferably, thepowder form of the yeast cells comprises greater than about 10⁷ to 10¹⁰yeast cells per gram.

5.1.4 Conditioning of Yeast Cells

According to the invention, performance of the activated yeast cells canbe optimized by culturing the activated yeast cells in the presence ofan extract from the stomach (e.g., the gastric juice) of an animal withphysiology similar to the subject to which the biological compositionwill be administered. The inclusion of this additional conditioningprocess allows the activated yeast cells to adapt to and endure theacidic environment of the subject's stomach. The method for conditioningactivated yeast cells of the invention comprises culturing yeast cellsin such materials in the presence of at least one EM field.

The culture process can be initiated by inoculating 1,000 ml of aconditioning medium with about 10 gram of dried activated yeastscontaining about 10¹⁰ cells per gram (as prepared by the methodsdescribed in Section 5.1.3). An equivalent number of yeast cells inculture, preferably greater than 10⁶ to 10⁹ cells per ml, morepreferably at 10⁸ cells per ml, can also be used as an inoculum. Theconditioning medium comprises per 1,000 ml about 700 ml of gastric juiceof an animal and about 300 ml of wild hawthorn juice. The process can bescaled up or down according to needs.

The gastric juice of an animal can be obtained from the stomach contentof a freshly slaughtered animal. Although not essential, the animal ispreferably kept under a clean environment, and fed a standard diet,preferably germ-free. For example, the content of the stomach of a120-day old pig is mixed with 2,000 ml of distilled water, and allowedto settle without stirring for 6 hours. The clear liquid above iscollected for use as the gastric juice used in the conditioning process.The gastric juice of a pig can be used to condition yeast cells for usein a variety of mammals, including humans. Other methods that can beused to collect the gastric juice include centrifugation or filtrationof the mixture to remove debris and/or microorganisms. The gastric juiceso obtained can be stored at 4° C. Preferably, the collection proceduresand storage are carried out under sterile conditions.

The wild hawthorn juice is an extract of wild hawthorn fruits preparedby slicing the fruits and drying the slices in air, preferably to lessthan 8% moisture (commercial dryer can be used if necessary), crushingthe dried fruits to less than 20 mesh, and mixing 1,500 ml of water per500 gram of the crushed wild hawthorn. The mixture is then allowed tosettle without stirring for 6 hours, and the clear liquid above iscollected for use as the wild hawthorn juice used in the conditioningprocess. Other methods that can be used to collect the hawthorn juiceinclude centrifugation or filtration of the mixture. Preferably, thecollection procedures and storage are carried out under sterileconditions.

The activated yeast cells are conditioned by culturing in at least oneof the following two EM fields which can be applied by the apparatusdescribed in Section 5.1.2 or any means known in the art:

The first EM field has a frequency in the range of 12,461 to 12,470 MHzand a field strength in the range of 300 to 320 mV/cm. The temperatureis maintained at 28° C. to 32° C., and typically at 30° C. The yeastculture is exposed to this first EM field for about 12 hours.

The second EM field has a frequency in the range of 12,761 to 12,770 MHzand a field strength in the range of 300 to 330 mV/cm. The temperatureis maintained at 28° C. to 32° C., and typically at 30° C. The yeastculture is exposed to this second EM field for about 32 hours.

In a preferred embodiment, the activated yeast cells are conditioned byculturing in both of the above-mentioned EM fields. In less preferredembodiments, the yeast cells are conditioned in the two different EMfields in a different order. In other embodiments, a series of EM fieldshaving field characteristics within the ranges stated above can beapplied to condition the yeast cells. The yeast cells can remain in thesame container and use the same set of electromagnetic wave generatorand emitters when switching from one EM field to another EM field.

The cell density of the culture at the end of the activation process istypically greater than about 10⁷ to 10¹⁰ cells per ml (estimated byhematocytometer). The activated and conditioned yeast cells may berecovered from the culture by various methods known in the art, andstored at a temperature below about 0° C. to 4° C.

The activated and conditioned yeast cells can be used directly in abiological composition or used as a starter culture for large scalemanufacturing. The activated and conditioned yeast cells recovered fromthe liquid culture may be dried and stored in powder form. Preferably,the powder form of the activated and conditioned yeast cells comprisesgreater than about 10⁸ to 10¹¹ yeast cells per gram.

5.1.5 Large Scale Manufacturing

The present invention also encompasses methods of manufacturing of thebiological compositions of the invention at a large scale. The activatedand conditioned yeast cells as prepared by Sections 5.1.3 and 5.1.4 arepropagated on a large scale to make the biological compositions of theinvention. The method comprises culturing the yeast cells in thepresence of one or more EM fields for a period of time, diluting thegrowing yeast cells with fresh medium, and repeating the process. Themethod can be carried out as a batch process or a continuous process.

In one preferred embodiment, a set of three containers (5, 6, 7) eachcomprising a set of electrodes for generating an electromagnetic fieldas described in Section 5.1.2 are set up each with 1,000 liters of aculture medium. See FIG. 2. The culture medium comprises nutrientsassimilable by the yeast cells as shown in Table 3.

TABLE 3 Material Quantity Wild hawthorn juice 300 liters Jujube juice300 liters Wu wei zi juice 300 liters Soybean juice 100 liters

The wild hawthorn juice is an extract of fresh wild hawthorn fruitsprepared by washing the fruits clean, drying the fruits in air or usinga commercial dryer to less than 8% moisture, crushing the dried fruitsto less than 20 mesh, and mixing the crushed wild hawthorn with water ata ratio of 400 liters of water per 100 kg of crushed fruits. The mixtureis then stirred continuously for 12 hours while the temperature ismaintained at 28° C. to 30° C. The mixture is then centrifuged at 1,000rpm to collect the supernatant which is used as described above.Preferably, the procedures are carried out under sterile conditions.

The jujube juice is an extract of fresh jujube fruits prepared bywashing the fruits clean, drying the fruits to less than 8% moisture,crushing the dried fruits to less than 20 mesh, and mixing the crushedjujube with water at a ratio of 400 liters of water per 100 kg ofcrushed fruits. The mixture is then stirred continuously for 12 hourswhile the temperature is maintained at 28° C. to 30° C. The mixture isthen centrifuged at 1,000 rpm to collect the supernatant which is usedas described above. Preferably, the procedures are carried out understerile conditions.

The wu wei zi juice is an extract of fresh berries of Schisandrachinensis plant prepared by washing the berries, drying the fruits toless than 8% moisture, crushing the dried berries to less than 20 mesh,and mixing the crushed berries with water at a ratio of 400 liters ofwater per 100 kg of crushed berries. The mixture is then stirredcontinuously for 12 hours while the temperature is maintained at 28° C.to 30° C. The mixture is then centrifuged at 1,000 rpm to collect thesupernatant which is used as described above. Preferably, the proceduresare carried out under sterile conditions.

The soybean juice is prepared by washing the soybeans, drying thesoybeans to less than 8% moisture, crushing the soybeans to less than 20mesh, and mixing the crushed soybeans with water. For 30 kg of soybeans,130 liters of water is used. The mixture is then stirred continuouslyfor 12 hours while the temperature is maintained at 28° C. to 30° C. Themixture is then centrifuged at 1,000 rpm to collect the supernatantwhich is used as described above. Preferably, the procedures are carriedout under sterile conditions.

The first container is inoculated with activated or activated andconditioned yeast cells as prepared by the methods of Sections 5.1.4 and5.1.5. About 1,000 gram of dried yeast powder are added to 1,000 literof culture medium. Each gram of the dried yeast powder comprises about10¹⁰ yeast cells. Instead of dried yeast cells, an equivalent number ofyeast cells in a liquid medium can also be used, preferably greater thanabout 10⁶ to 10⁹ cells per ml, more preferably about 10⁷ cells per ml.

The yeast cells in the first container (5) are then subjected to aseries of two EM fields. For the first EM field, which can be applied bythe apparatus described in Section 5.1.2, the frequency is in the rangeof 12,461 to 12,470 MHz and the field strength is in the range of 310 to330 mV/cm. The yeast culture is exposed to this first EM field for about8 hours. The yeast cells are then subjected to a second EM field havinga frequency in the range of 12,761 to 12,770 MHz and a field strength inthe range of 330 to 350 mV/cm. The yeast culture is exposed to thissecond EM field for about 12 hours. The yeast cells from the firstcontainer are then transferred to the second container which containsabout 1,000 liter of the culture medium. In effect, the first yeastculture is diluted by about 50% with fresh culture medium.

In the second container (6), the yeast cells are again subjected to aseries of two EM fields. The frequencies used in the second containerare similar to those used in the first container but the field strengthsare marginally lower. The first EM field has a frequency in the range of12,461 to 12,470 MHz and a field strength in the range of 330 to 340mV/cm. The yeast culture is exposed to this EM field for about 8 hours.The yeast cells are then subjected to a second EM field having afrequency in the range of 12,761 to 12,770 MHz and a field strength inthe range of 350 to 370 mV/cm. The yeast culture is exposed to thissecond EM field for about 12 hours. The yeast cells from the secondcontainer are then transferred to the third container which contains yetanother 1,000 liter of the culture medium. Again, the second yeastculture is diluted by about 50% with fresh culture medium.

In the third container (7), the yeast cells are again subjected to aseries of two EM fields. The frequencies used in the third container aresimilar to those used in the first and second container but the fieldstrengths are lower. The first EM field has a frequency in the range of12,461 to 12,470 MHz and field strength in the range of 220 to 240mV/cm. The yeast culture is exposed to this EM field for about 12 hours.The yeast cells are then subjected to a second EM field having afrequency in the range of 12,761 to 12,770 MHz and a field strength inthe range of 250 to 270 mV/cm. The yeast culture is exposed to this EMfield for about 24 hours.

The yeast cell culture resulting from the end of this stage can be useddirectly as an oral composition of the invention, or used to form othercompositions encompassed by the invention.

The cell density of the culture at the end of the large scalemanufacturing process is typically greater than about 10⁸ to 10¹⁰ cellsper ml (estimated by hematocytometer). The concentration of yeast cellsin the medium can be concentrated or diluted accordingly. In certainembodiments, the concentration of yeast cells in the medium is in therange of 10³ to 10¹⁰ cells per ml. In less preferred embodiments, theconcentration of yeast cells in the medium is in the range of 10³ to 10⁶cells per ml. In more preferred embodiments, the concentration of yeastcells in the medium is greater than 10⁶ to 10¹⁰ cells per ml. In mostpreferred embodiments, the concentration of yeast cells in the medium isin the range of 10⁶ to 5×10⁸ cells per ml.

Other ingredients that enhance the healthful benefits, pharmacologicalproperties and/or organoleptic characteristics of the composition can beadded to the yeast cell culture. To maintain viability and freshness ofthe composition, it is preferred that the various downstream andpackaging process be carried out below room temperature, and preferablyat 0° C. to 4° C. In one embodiment, the yeast cell culture can bepackaged in liquid containers.

In another embodiment, the activated and conditioned yeast cells can bedried as follows. The yeast cell culture is first centrifuged under 75to 100 g for 10 to 20 minutes to remove the supernatant. The residuewhich may contain up to 85% moisture is dried in a first dryer at atemperature not exceeding 60±2° C. for a period of 5 minutes so thatyeast cells quickly became dormant. The yeast cells were then sent to asecond dryer and dried at a temperature not exceeding 65±2° C. for aperiod of about 8 minutes to further remove at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 98%, or at least 99% ofwater. For example, the yeast cells may be dried to remove at least 88%of water so the dried yeast cells may contain up to 12% moisture.

After cooling to room temperature, the dried yeast cells can be packagedby standard pharmaceutical methods in various solid dosage form, eachcontaining a predetermined amount of the dried material. In a preferredembodiment, the dried material comprises about 10⁵ to 10¹¹ cells pergram. In a more preferred embodiment, the dried material comprises about10⁸ to 5×10¹⁰ cells per gram. In a most preferred embodiment, the driedmaterial comprises about 5×10⁸ cells per gram.

In general, the compositions are prepared by uniformly and intimatelyadmixing the active ingredient with liquid carriers or finely dividedsolid carriers.

5.1.6 Preferred Embodiments

In one preferred embodiment, the invention provides a method forpreparing a biological composition comprising activated and conditionedyeast cells, said method comprising in any order the steps of:

-   (a) culturing the yeast cells in a first electromagnetic field    having a frequency at 7,967 MHz and a field strength of 264 mV/cm;-   (b) culturing the yeast cells in a second electromagnetic field    having a frequency at 10,188 MHz and a field strength of 266 mV/cm;-   (c) culturing the yeast cells in a third electromagnetic field    having a frequency at 12,281 MHz and a field strength of 279 mV/cm;-   (d) culturing the yeast cells in a fourth electromagnetic field    having a frequency at 12,466 MHz and a field strength of 286 mV/cm;    and-   (e) culturing the yeast cells in a fifth electromagnetic field    having a frequency at 12,764 MHz and a field strength of 306 mV/cm;    and after the last of the first five steps, the following steps in    any order:-   (f) culturing the yeast cells in a liquid medium comprising wild    hawthorn juice and gastric juice of a mammal in a sixth    electromagnetic field having a frequency at 12,466 MHz and a field    strength of 306 mV/cm; and-   (g) culturing the yeast cells in a liquid medium comprising wild    hawthorn juice and gastric juice of a mammal in a seventh    electromagnetic field having a frequency at 12,764 MHz and a field    strength of 323 mV/cm.

The activated and conditioned yeast cells obtained at the conclusion ofthis method is encompassed by the invention. Preferably, the yeast cellsare Saccharomyces cerevisiae Hansen strain IFFI 1413. These yeast cellscan be used in the following method of further expanding number ofactivated and conditioned yeast cells.

In another preferred embodiment, the invention provides a method of massproducing a biological composition comprising activated and conditionedyeast cells, said method comprising culturing the activated andconditioned yeast cells prepared by the preferred embodiment describedabove in this section, in a medium comprising wild hawthorn juice,jujube juice, wu wei zi juice, and soybean juice, and in the presence ofone or more series of electromagnetic fields. Each series of EM fieldscomprises two EM fields in the order stated:

-   (h) an eighth electromagnetic field or series of electromagnetic    fields having a frequency at 12,466 MHz and a field strength in the    range of 220 to 340 mV/cm, preferably at three fields strengths,    e.g., in the order of 328 mV/cm, 334 mV/cm, and 238 mV/cm; and-   (i) a ninth electromagnetic field or series of electromagnetic    fields having a frequency at 12,764 MHz and a field strength in the    range of 250 to 370 mV/cm, preferably at three fields strengths,    e.g., in the order of 338 mV/cm, 362 mV/cm, and 263 mV/cm.

The series may be repeated several times, such as three times, each timeusing a slightly lower field strength.

5.2 Methods of Uses

5.2.1 Uses In Subjects with Pancreatic Cancer

The present invention further provides methods of use of the biologicalcompositions of the invention. In one embodiment, the biologicalcomposition is used as a medicament for treatment of pancreatic cancer.In another embodiment, the biological composition is used as a dietarysupplement, health food, or health drink. The methods compriseadministering an effective amount of the biological composition to asubject in need. The biological composition may be administered orally,in liquid or solid form, or enterally through a feeding tube. As usedherein, the term “an effective amount” means an amount sufficient toprovide a therapeutic or healthful benefit in the context of pancreaticcancer.

According to the invention, the biological composition can produce ahealthful benefit in a subject suffering from pancreatic cancer.Preferably, the subject is a human being. The subject in need is one whois diagnosed with pancreatic cancer, with or without metastasis, at anystage of the disease (e.g., TX, T0, Tis, T1, T2, T3, T4, NX, N0, N1, MX,M0 and M1). As used herein, the term “pancreatic cancer” includes but isnot limited to adenocarcinomas, acinar cell carcinoma,cystadenocarcinoma (mucinous), adenosquamous carcinoma, solidmicroglandular carcinoma, carcinoid, sarcoma, and malignant lymphoma.

The subject may be a pancreatic cancer patient who is receivingconcurrently other treatment modalities against the pancreatic cancer.The subject can be a pancreatic cancer patient who had undergone aregimen of treatment (e.g., chemotherapy and/or radiation) and whosecancer is regressing. The subject may be a pancreatic cancer patient whohad undergone a regimen of treatment (e.g., surgery) and who appears tobe clinically free of the pancreatic cancer. The biological compositionof the invention can be administered adjunctively with any of thetreatment modalities, such as but not limited to chemotherapy,radiation, and/or surgery. For example, the biological composition canbe used in combination with one or more chemotherapeutic orimmunotherapeutic agents, such as hexamethylmelamine, bleomycin,cisplatin, mitomycin C, doxorubicin, methotrexate and Gemzar(gemcitabine HCL). The biological composition can also be used afterother regimen(s) of treatment is concluded.

The subject may be one who has not yet been diagnosed with pancreaticcancer but are predisposed to or at high risk of developing pancreaticcancer as a result of genetic factors and/or environmental factors. Thesubject may also be one who displays characteristics that are associatedwith a high risk of pancreatic cancer, such as nodules detected bycomputer tomographic scanning or suspect cells in biopsy and/or bodyfluids.

Depending on the subject, the therapeutic and healthful benefits rangefrom inhibiting or retarding the growth of the pancreatic cancer and/orthe spread of the pancreatic cancer to other parts of the body (i.e.,metastasis), palliating the symptoms of the cancer, improving theprobability of survival of the subject with the cancer, prolonging thelife expectancy of the subject, improving the quality of life of thesubject, and/or reducing the probability of relapse after a successfulcourse of treatment (e.g., surgery, chemotherapy or radiation). Thesymptoms associated with pancreatic cancer include abdominal pain,unexpected weight loss, nausea, loss of appetite, weight loss, digestiveproblems, jaundice, or yellowing of the skin, restlessness, loss ofenergy, irritability, sweating, tremor, drowsiness and severe confusion.

In particular, the invention provides a method for retarding the growthof pancreatic cancer cells in a subject, such as a human, comprisingadministering orally to the subject a biological composition of theinvention. The invention also provide a method for prolonging the timeof survival of a subject inflicted with pancreatic cancer, preferably ahuman patient, comprising administering orally to the subject abiological composition of the invention.

The effective dose will vary with the subject treated. The effectivedose for the subject will also vary with the condition to be treated andthe severity of the condition to be treated. The dose, and perhaps thedose frequency, will also vary according to the age, body weight, andresponse of the individual subject. In general, the total daily doserange of activated and conditioned yeast cells for a subject inflictedwith pancreatic cancer is from about 10⁵ to 10¹¹ cells per day;preferably, about 10⁸ to 5×10 cells per day; more preferably, about2×10⁹ cells per day in powder form or 9×10⁸ to 1×10¹⁰ cells per day inliquid preparations, administered in single or divided doses orally. Thelength of time for a course of treatment should be at least 1 week, atleast 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, atleast 7 weeks, at least 10 weeks, at least 13 weeks, at least 15 weeks,at least 20 weeks, at least 6 months, or at least 1 year. It may benecessary to use dosages outside these ranges in some cases as will beapparent to those skilled in the art. In certain embodiments, the oralcompositions can be administered for a period of time until the symptomsand/or infection of the patients by the bacteria and viruses are undercontrol, or when the disease has regressed partially or completely. Foruse as a dietary supplement, the total daily dose range should be fromabout 10⁵ to 10¹¹ cells per day; preferably, about 5×10⁷ to 5×10⁹ cellsper day. The oral compositions can be administered as a dietarysupplement for as long as 6 months, or in accordance with recommendedlength of use under the Dietary Supplement Health and Education Act(DSHEA) or other government or industry guidelines. Further, it is notedthat the nutritionist, dietician, clinician or treating physician willknow how and when to interrupt, adjust, or terminate use of thebiological composition as a medicament or dietary supplement inconjunction with individual patient response.

The effect of the biological compositions of the invention ondevelopment and progression of pancreatic cancer can be monitored by anymethods known to one skilled in the art, including but not limited tomeasuring: a) changes in the size and morphology of the tumor usingimaging techniques such as a computed tomographic (CT) scan or asonogram; and b) changes in levels of biological markers of risk forpancreatic cancer.

5.2.2 Formulations

The biological compositions of the present invention comprise activatedand conditioned live yeast cells prepared as described above in Section5.1, as active ingredient, and can optionally contain a pharmaceuticallyacceptable carrier or excipient, and/or other ingredients provided thatthese ingredients do not kill or inhibit the yeast cells. Otheringredients that can be incorporated into the biological compositions ofthe present invention, may include, but are not limited to, herbs(including traditional Chinese medicine products), herbal extracts,vitamins, amino acids, metal salts, metal chelates, coloring agents,flavor enhancers, preservatives, and the like.

Any dosage form may be employed for providing the subject with aneffective dosage of the oral composition. Dosage forms include tablets,capsules, dispersions, suspensions, solutions, and the like. In oneembodiment, compositions of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets, or tablets, each containing a predetermined amount of activatedand conditioned yeast cells, as a powder or granules or as a solution ora suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-wateremulsion, or a water-in-oil liquid emulsion. In general, thecompositions are prepared by uniformly and intimately admixing theactive ingredient with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product into the desiredpresentation. Such products can be used as pharmaceuticals or dietarysupplements, depending on the dosage and circumstances of its use.

The oral compositions of the present invention may additionally includebinding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); binders or fillers (e.g., lactose,pentosan, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets or capsules can be coated bymethods well known in the art.

Liquid preparations for oral administration can take the form of, forexample, solutions, syrups or suspensions, or they can be presented as adry product for constitution with water or other suitable vehicle beforeuse. The temperature of the liquid used to reconstitute the driedproduct should be less than 65° C. Such liquid preparations can beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, cellulosederivatives or hydrogenated edible fats); emulsifying agents (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oilyesters, ethyl alcohol or fractionated vegetable oils); and preservatives(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). As describedbelow, the preparations can also be made to resemble foods or beverages,containing buffer salts, flavoring, coloring and sweetening agents asappropriate. In certain embodiments, the oral composition is a cellsuspension comprising about 10³ to 10¹⁰ cells per ml. The oralcomposition can be produced by diluting or concentrating the yeastculture medium produced by the method of Section 5.1.5 as required. Inless preferred embodiments, the oral composition is a cell suspensioncontaining about 10³ to 10⁶ cells per ml. In more preferred embodiments,the oral composition is a cell suspension containing greater than about10⁶ to 10¹⁰ cells per ml. In most preferred embodiments, the oralcomposition is a cell suspension containing about 10⁶ to 5×10⁸ cells perml. The oral composition can be formulated as a health drink andpackaged in liquid containers, each containing a predetermined amount ofthe liquid yeast culture. Standard methods of quality control andpackaging are applied to produce in one embodiment of the invention,oral compositions packaged in liquid containers each comprising about 1ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 15 ml, 20 ml, 30 ml, 40 ml, 50 ml, 75ml, 100 ml, 150 ml, 200 ml, 250 ml, 500 ml, 750 ml, or 1,000 ml of thelive yeast cells. The number of container to be taken each day to obtainthe total daily dose in a subject depends on the number of activated andconditioned yeast cells contained within each container. For example, acontainer may comprise 50 ml of liquid with 10⁷ cells per ml and when atotal daily dose of about 2×10⁹ cells per day is desired, a subject candrink 4 containers per day to obtain the desired total daily dose.

Generally, because of their ease of administration, tablets and capsulesrepresent the most advantageous oral dosage unit form, in which casesolid pharmaceutical carriers as described above are employed. In apreferred embodiment, the composition is a capsule. The capsules can beformulated by any commercially available methods. In certainembodiments, the composition is a capsule containing 5 mg, 10 mg, 15 mg,20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 300mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1.0 gram, 1.25 gram,1.5 gram, or 2.0 gram of live yeast cells in powder form. The powder inthe capsule comprises about 10⁵ to about 10¹¹ cells per gram; morepreferably, about 10⁸ to 5×10¹⁰ cells per gram; and most preferably,about 5×10⁸ cells per gram. The number of capsule to be taken each dayto obtain the total daily dose in a subject depends on the number ofactivated and conditioned yeast cells contained within each capsule. Forexample, a capsule may comprise about 500 mg of powder with 5×10⁸ cellsper gram. To achieve a total daily dose of about 2×10⁹ cells per day, asubject can take two capsules at a time for four times per day.

In another embodiment, the biological compositions comprising activatedand conditioned yeast cells can be added directly to foods so that aneffective amount of yeast cells is ingested during normal meals. Anymethods known to those skilled in the art may be used to add to orincorporate the biological compositions into natural or processed foods,provided that the activated and conditioned yeast cells remain viable.Preferably, the nutritional compositions of the invention are made andstored under conditions, such as temperature, from about 0° C. to 4° C.As used herein, the term “food” broadly refers to any kind of material,liquid or solid, that is used for nourishing an animal, and forsustaining normal or accelerated growth of an animal including humans.Many types of food products or beverages, such as but not limited to,fruit juice, herbal extracts, tea-based beverages, dairy products,soybean product (e.g., tofu), and rice products, can be used to formnutritional compositions comprising the activated and conditioned yeastcells of the invention.

The invention is further defined by reference to the following exampledescribing in detail the animal trials conducted to study the efficacyand safety of activated and conditioned yeast cells of the invention.

6. EXAMPLE

The following example illustrates the benefit of a biologicalcomposition of the invention in a BALB/c mouse model of human pancreaticcancer. The growth of tumors in the mice was studied.

Numerous animal studies have reported the use of BALB/c mice in thestudy of treating pancreatic cancer. The use of orthotopic transplanttechnique has been highly successful in the development of murine modelsof human pancreatic cancer.

The human pancreatic cancer sample is carefully selected before, during,and after surgery in order to ensure transplant occurred at theequivalent position in the mouse. The pancreatic carcinoma sample isobtained from patients who have not received any radiation, chemotherapyor immune enhancement treatment.

The biological composition comprising 10⁸ cells per ml of activated andconditioned yeast cells of the strain Saccharomyces cerevisiae Hansenstrain IFFI 1413 was prepared by the methods described in Section 5.1and subsections therein.

6.1 Animal Preparation

The animals used to generate the pancreatic cancer cells for theexperiments were BALB/c mice, both male and female with an average bodyweight of about 18 to 20 gram (obtainable from the Chinese Academy ofMilitary Medical Sciences, Beijing, China). The mice were starved for 24hours before the surgery. The pancreatic tumor cells were isolated fromclinical biopsy samples in 1987 (obtainable from the Cancer Institute,Chinese Academy of Medical Sciences, Beijing, China).

The animals were starved for 24 hours before the experiment. Asuspension containing about 1×10⁶ pancreatic cancer tumor cells (about0.2 ml culture suspension) was transplanted into the donors animals atthe thorax by injection. Animals that showed robust growth of the tumorwere used.

6.2 Experimental Design

The mice injected with tumor cells were divided into 4 experimentalgroups of ten mice per group and one control group. The fourexperimental groups were triplicated (i.e., using a total of 120 mice inthe experimental groups). In group AY, the mice received 0.3 ml of thebiological composition once per day. In group NY, the mice received 0.3ml of the untreated yeast cells once per day. In group VDS, the micewere injected intravenously with 3 mg of vindesine (VDS) per kg bodyweight once a week for four weeks. In group CK1, the mice received 0.3ml of physiological saline once per day. A fifth group of mice, groupCK2, which did not receive tumor cells, was given 0.3 ml ofphysiological saline per day.

The mice received the biological compositions, untreated yeast cells,VDS or saline three days after the tumor cells were transplanted. Themice in group CK2 also started receiving saline on the same day as theother four groups. The biological compositions, untreated yeast cellsand saline were administered orally by a feeding tube and the VDS byintravenous injection for 30 consecutive days. On the 31^(st) day fromtumor inoculation, the mice were sacrificed and the weight of the miceas well as the weight of the tumor were determined by standardtechniques.

6.3 Results

Table 4 shows the differences in the weight of the mice and tumors ofthe mice in the various treatment and control groups.

TABLE 4 mean weight of tumor mean weight of mice and nodules andstandard Group standard deviation (g) deviation (mg) AY 19.8 ± 2.5 0.44± 0.3 NY 17.7 ± 3.2 2.86 ± 0.9 MMC 18.9 ± 3.4 1.37 ± 0.7 CK1 17.6 ± 3.62.84 ± 0.9 CK2 20.3 ± 2.4 not applicable

The mice bearing pancreatic cancer cells that received 0.3 ml of thebiological composition of the invention (group AY) showed the leastdeviation in the body weight and weight of esophageal as compared tohealthy mice not injected tumor cells (group CK2). The mice in group AYalso had less tumor mass as compared to mice that did not receivetreatment (group CK1) as well as the mice in group NY (0.3 ml ofuntreated yeast cells per day) and the mice in group VDS (3 mg ofvindesine per kg body weight per week).

7. EXAMPLE

The following example illustrates the benefit of a biologicalcomposition of the invention in a BALB/c mouse model of human pancreaticcancer. The survival time of mice after tumor injection and treatmentwas studied.

The biological composition comprising 10⁸ cells per ml of activated andconditioned yeast cells of the strain Saccharomyces cerevisiae Hansenstrain IFFI 1413 was prepared by the methods described in Section 5.1and subsections therein.

7.1 Animal Preparation

The animals were prepared in a similar manner as described in Section6.1.

7.2 Experimental Design

The mice injected with tumor cells were divided into 4 experimentalgroups of ten mice per group and one control group. The fourexperimental groups were triplicated (i.e., using a total of 120 mice inthe experimental groups). In group 2AY, the mice received 0.3 ml of thebiological composition once per day. In group 2NY, the mice received 0.3ml of the untreated yeast cells once per day. In group 2VDS, the micewere injected intravenously with 3 mg of vindesine (VDS) per kg bodyweight once a week for four weeks. In group 2CK1, the mice received 0.3ml of physiological saline once per day. A fifth group of mice, group2CK2, which did not receive tumor cells, was given 0.3 ml ofphysiological saline per day.

The mice received the biological compositions, untreated yeast cells,VDS or saline on the same day as the tumor cells were transplanted. Themice in group 2CK2 also started receiving saline on the same day as theother four groups. The biological compositions, untreated yeast cellsand saline were administered orally by a feeding tube and the VDS byintravenous injection for 30 consecutive days. The mice were observedover 6 months from the day of tumor inoculation and survival wasrecorded. The weight of the mice as well as the weight of the tumor weredetermined by standard techniques.

7.3 Results

Table 5 shows the number of mice in the various treatment and controlgroup that survived the tumor injection over a period of 6 months. Eachof the 30 mice in each group received 30 consecutive days of eitheruntreated yeast cells, VDS, saline or biological compositions of theinvention. Table 6 shows the weight of the mice that survived and theweight of their tumors in the various treatment and control groups.

TABLE 5 Number of live animals remaining in the groups after 30 days oftreatment Time after cessation of Group Group Group Group Grouptreatment 2AY 2NY 2VDS 2CK1 2CK2 0 month 30 30 30 28 30 1 month 30 27 3022 30 2 months 30 17 27 6 30 3 months 30 0 27 0 30 4 months 29 0 11 0 305 months 29 0 0 0 30 6 months 29 0 0 0 30

TABLE 6 mean weight of mice mean weight of tumor and standard nodulesand standard Group deviation (g) deviation (mg) 2AY 20.7 ± 3.5 78.3 ±14.2 2NY all animals dead all animals dead 2VDS all animals dead allanimals dead 2CK1 all animals dead all animals dead 2CK2 21.3 ± 2.6 notapplicable

The mice bearing pancreatic cancer cells that received 0.3 ml of thebiological composition of the invention (group 2AY) survived for morethan 6 months and the tumor never reoccurred. On the contrary, the micein group 2NY (0.3 ml of untreated yeast cells per day), group 2VDS (3 mgof vindesine per kg body weight per week) and group 2CK1 (0.3 ml ofsaline per day) all died after four months from injection of tumorcells.

As in Example 6, the mice bearing pancreatic cancer cells that received0.3 ml of the biological composition of the invention (group 2AY) showedthe least deviation in the weight of mice as compared to healthy micenot injected tumor cells (group 2CK2).

8. EXAMPLE

The following example illustrates the benefit of a biologicalcomposition of the invention in a kun ming mouse model of humanpancreatic cancer. The growth of tumors in the mice was studied.

Numerous animal studies have reported the use of murine models in thestudy of treating pancreatic cancer. There is a 100% success rate fortransplanting mice with the pancreatic tumor type MPC-83. Detaileddescription of the transplantable mouse pancreatic cancer cell lineMPC-83 can be found in Hu M. Y., 1968, Zhonghua Zhong Liu Za Zhi(Chinese) 8(1):1–3, which is incorporated herein by reference in itsentirety.

The biological composition comprising 10⁸ cells per ml of activated andconditioned yeast cells of the strain Saccharomyces cerevisiae Hansenstrain IFFI 1413 was prepared by the methods described in Section 5.1and subsections therein.

8.1 Animal Preparation

The animals used for the experiments were kun ming mice, 6 to 8 weeksold (obtainable from the Chinese Academy of Military Medical Sciences,Beijing, China). Both male and females with an average weight of about15 to 18 gram were used. The transplantable mouse pancreatic cancer cellline MPC-83 (obtainable from Kun-Ming Medical University, Kun-Ming,China) in a suspension containing about 1.2×10⁷ viable tumor cells(about 0.2 ml culture suspension) was injected subcutaneously into theanimals.

8.2 Experimental Design

The mice injected with tumor cells were kept for 5 days and were dividedinto 4 experimental groups of ten mice per group and one control group.The four experimental groups were triplicated (i.e., using a total of120 mice in the experimental groups). In group AY, the mice received 0.3ml of the biological composition once per day. In group NY, the micereceived 0.3 ml of the untreated yeast cells once per day. In group MMC,the mice were injected intravenously with 10⁵ units of mitomycin C (MMC)per kg body weight per day. In group CK1, the mice received 0.3 ml ofphysiological saline once per day. A fifth group of mice, group CK2,which did not receive tumor cells, was given 0.3 ml of physiologicalsaline per day.

The mice received the biological compositions, untreated yeast cells,MMC or saline on the same day as the tumor cells were transplanted. Themice in group CK2 also started receiving saline on the same day as theother four groups. The biological compositions, untreated yeast cellsand saline were administered orally by a feeding tube and the MMC byintravenous injection for 30 consecutive days. On the 31^(st) day fromtumor inoculation, the mice were sacrificed and the weight of the miceas well as the weight of the tumor were determined by standardtechniques.

8.3 Results

Table 7 shows the differences in the weight of the mice and tumors ofthe mice in the various treatment and control groups.

TABLE 7 mean weight of tumor mean weight of mice and nodules andstandard Group standard deviation (g) deviation (g) AY 19.2 ± 2.2 0.9 ±0.4 NY 17.4 ± 2.4 3.3 ± 1.5 MMC 18.3 ± 1.5 2.8 ± 1.2 CK1 17.6 ± 2.4 3.6± 1.7 CK2 20.3 ± 2.3 not applicable

The mice bearing pancreatic cancer cells that received 0.3 ml of thebiological composition of the invention (group AY) showed the leastdeviation in the weight of mice as compared to healthy mice not injectedtumor cells (group CK2). The mice in group AY also had less tumor massas compared to mice that did not receive treatment (group CK1) as wellas the mice in group NY (0.3 ml of untreated yeast cells per day) andthe mice in group MMC (10⁵ units of mitomycin C per kg body weight perday).

9. EXAMPLE

The following example illustrates the benefit of a biologicalcomposition of the invention in a kun ming mouse model of humanpancreatic cancer. The survival time of mice after tumor injection andtreatment was studied.

The biological composition comprising 10⁸ per ml of activated andconditioned yeast cells of the strain Saccharomyces cerevisiae Hansenstrain IFFI 1413 was prepared by the methods described in Section 5.1and subsections therein.

9.1 Animal Preparation

The animals were prepared in a similar manner as described in Section8.1.

9.2 Experimental Design

The mice injected with tumor cells were kept for 5 days and were dividedinto 4 experimental groups of ten mice per group and one control group.The four experimental groups were triplicated (i.e., using a total of120 mice in the experimental groups). In group 2AY, the mice received0.5 ml of the biological composition once per day. In group 2NY, themice received 0.5 ml of the untreated yeast cells once per day. In group2MMC, the mice were injected intravenously with 1.5×10⁵ units ofmitomycin C (MMC) per kg body weight per day. In group 2CK1, the micereceived 0.5 ml of physiological saline once per day. A fifth group ofmice, group 2CK2, which did not receive tumor cells, was given 0.5 ml ofphysiological saline per day.

The mice received the biological compositions, untreated yeast cells,MMC or saline on the same day as the tumor cells were transplanted. Themice in group 2CK2 also started receiving saline on the same day as theother four groups. The biological compositions, untreated yeast cellsand saline were administered orally by a feeding tube and the MMC byintravenous injection for 30 consecutive days. The mice were observedover 6 months from the day of tumor inoculation and survival wasrecorded. The weight of the mice as well as the weight of the tumor weredetermined by standard techniques.

9.3 Results

Table 8 shows the number of mice in the various treatment and controlgroup that survived the tumor injection over a period of 6 months. Eachof the 30 mice in each group received 30 consecutive days of eitheruntreated yeast cells, MMC, saline or biological compositions of theinvention. Table 9 shows the weight of the mice that survived and theweight of their tumors in the various treatment and control groups.

TABLE 8 Number of live animals remaining in the groups after 30 days oftreatment Time after cessation of Group Group Group Group Grouptreatment 2AY 2NY 2MMC 2CK1 2CK2 0 month 30 27 30 24 30 1 month 30 0 270 30 2 months 30 0 21 0 30 3 months 30 0 7 0 30 4 months 30 0 0 0 30 5months 30 0 0 0 30 6 months 30 0 0 0 30

TABLE 9 mean weight of tumor mean weight of mice and nodules andstandard Group standard deviation (g) deviation (mg) 2AY 20.5 ± 1.6104.1 ± 16.7 2NY all animals dead all animals dead 2MMC all animals deadall animals dead 2CK1 all animals dead all animals dead 2CK2 21.3 ± 2.6not applicable

The mice bearing pancreatic cancer cells that received 0.5 ml of thebiological composition of the invention (group 2AY) survived for morethan 6 months and the tumor never reoccurred. On the contrary, the micein group 2NY (0.5 ml per day of untreated yeast cells), group 2MMC(1.5×10⁵ units of mitomycin C per kg body weight per day) and group CK1(0.5 ml of saline per day) all died after four months from injection oftumor cells.

As in Example 7, the mice bearing pancreatic cancer cells that received0.5 ml of the biological composition of the invention (group 2AY) showedthe least deviation in the weight of mice as compared to healthy micenot injected tumor cells (group 2CK2).

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein, will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

1. A method of treating yeast cells, comprising at least two stepsselected from the group consisting of: a. culturing yeast cells in afirst electromagnetic field having a frequency in the range of 7,961 to7,970 MHz and a field strength in the range of 250 to 270 mV/cm; b.culturing the yeast cells in a second electromagnetic field having afrequency in the range of 10,181 to 10,190 MHz and a field strength inthe range of 250 to 270 mV/cm; c. culturing the yeast cells in a thirdelectromagnetic field having a frequency in the range of 12,276 to12,285 MHz and a field strength in the range of 270 to 290 mV/cm; d.culturing the yeast cells in a fourth electromagnetic field having afrequency in the range of 12,461 to 12,470 MHz and a field strength inthe range of 280 to 300 mV/cm; and e. culturing the yeast cells in afifth electromagnetic field having a frequency in the range of 12,761 to12,770 MHz and a field strength in the range of 300 to 320 mV/cm, andwherein said method further comprises the step of recovering thecultured yeast cells.
 2. A method of treating yeast cells, comprising atleast two steps selected from the group consisting of: a. culturingyeast cells in a first electromagnetic field having a frequency in therange of 7,961 to 7,970 MHz and a field strength in the range of 250 to270 mV/cm; b. culturing the yeast cells in a second electromagneticfield having a frequency in the range of 10,181 to 10,190 MHz and afield strength in the range of 250 to 270 mV/cm; c. culturing the yeastcells in a third electromagnetic field having a frequency in the rangeof 12,276 to 12,285 MHz and a field strength in the range of 270 to 290mV/cm; d. culturing the yeast cells in a fourth electromagnetic fieldhaving a frequency in the range of 12,461 to 12,470 MHz and a fieldstrength in the range of 280 to 300 mV/cm; and e. culturing the yeastcells in a fifth electromagnetic field having a frequency in the rangeof 12,761 to 12,770 MHz and a field strength in the range of 300 to 320mV/cm, wherein said method further comprising at least one step selectedfrom the group consisting of: f. culturing the yeast cells in a liquidmedium comprising wild hawthorn fruit juice and gastric juice of amammal in a sixth electromagnetic field having a frequency in the rangeof 12,461 to 12,470 MHz and a field strength in the range of 300 to 320mV/cm; and g. culturing the yeast cells in a liquid medium comprisingwild hawthorn fruit juice and gastric juice of a mammal in a seventhelectromagnetic field having a frequency in the range of 12,761 to12,770 MHz and a field strength in the range of 300 to 330 mV/cm, andwherein said method further comprises the step of recovering thecultured yeast cells.
 3. The method of claim 2, further comprisingbefore the recovering step the following steps, which can be carried outin any order: h. culturing the yeast cells in a first liquid medium andan eighth electromagnetic field or series of electromagnetic fieldshaving a frequency in the range of 12,461 to 12,470 MHz and a fieldstrength in the range of 220 to 340 mV/cm; and i. culturing the yeastcells in a second liquid medium and a ninth electromagnetic field orseries of electromagnetic fields having a frequency in the range of12,761 to 12,770 MHz and a field strength in the range of 250 to 370mV/cm, wherein the first and second liquid mediums each comprises wildhawthorn fruit juice, jujube fruit juice, wu wei zi berry juice, andsoybean juice.
 4. The method of claim 1, wherein the yeast cells to betreated are cells of Saccharomyces.
 5. The method of claim 1, whereinthe yeast cells to be treated are cells of Saccharomyces cerevisiae IFFI1413.
 6. The method of claim 2 or 3, wherein the yeast cells to betreated are cells of Saccharomyces.
 7. The method of claim 2 or 3,wherein the yeast cells to be treated are cells of Saccharomycescerevisiae IFFI
 1413. 8. The method of claim 2 or 3, further comprisingbefore the recovering step the following steps: j. drying the yeastcells at a temperature not exceeding 65° C. for a period of time,wherein the yeast cells become dormant; and k. drying the yeast cells ata temperature not exceeding 70° C. for a period of time to reduce themoisture content to below 5%.